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The key focus in this project was put on the characterization of the regulatory elements that our switch comprises of; repressor and anti-repressor from the Gifsy1 and Gifsy2 phages and terminator and anti-terminator from lambda and P22 phages.
As a proof of concept for the regulatory systems several constructs were made in low-copy-number plasmids. By means of the reporter proteins GFP and RFP we were able to investigate our colonies under a microscope and in a fluorometer to see if the expected reporters were expressed and thereby if the BioBricks work as expected. In addition to that, we also ran some measurements in a BioLector which can detect both OD and fluorescence simultaneously and thus also makes it probable that our BioBricks work as expected.
The measurements carried out in the BioLector also form the basis of the characterization of the BioBricks. From the plots derived from the measurements we are able to see the effect of the Gifsy1 and Gifsy2 promoters when the repressor is expressed.
As a means of characterizing the anti-terminator N we developed a Synthetic Promoter Library (SPL) which is an ideal way of fine-tuning gene expression. The SPL comprises of a wide variety of promoters with different promoter strengths and by cloning the SPL in front of the N protein it is possible to determine the right level of expression and thus the right promoter. However, as the idea for the SPL was evolved it became clear that the SPL could indeed be used to characterize and/or fine-tune other BioBricks and thus the idea of making the SPL as a standard was developed.
The experimental work has been divided into three parts. Here you can find results and characterization of: